Glucose transport proteins (GLUTs) catalyze equilibrative, cellular sugar transport, are essential for carbohydrate homeostasis in humans, contain 12 membrane-spanning alpha helices (TMs) which form a translocation pathway (amphipathic TMs 1, 2, 4, 5, 7, 8, 10 and 11) stabilized by a scaffold (hydrophobic TMs 3, 6, 9 and 12) and adopt 3 conformational states: the inward, the occluded and the outward orientations. Gaps in our understanding of the GLUTs are: 1) How substrates bind; 2) How substrates are translocated; 3) How GLUTs oligomerize. We address these gaps and ask how each process is altered in a human disease - GLUT1 deficiency syndrome (GLUT1-DS). Specific Aim 1 tests the hypothesis that substrates bind in the GLUT translocation pathway. GLUT1 transports glucose but not fructose while GLUT5 transports fructose not glucose. Mutagenesis studies suggest that TMs 2, 7 and 11 contribute to GLUT5 fructose binding and TMs 5, 7, 8, 10 and 11 to GLUT1 glucose binding. Using homology scanning mutagenesis, we ask if substitution of specific GLUT5 translocation TMs into GLUT1 converts GLUT1 from a glucose to a fructose transporter and vice versa and we ask which residues are critical for binding. Mass spectrometry of purified human GLUT1 will reveal which GLUT1 residues are modified by photo-affinity substrates/antagonists and which are substrate- protected against hydrogen-deuterium exchange (H/DX)? We then ask if critical substrate binding residues are altered in GLUT1-DS. Specific Aim 2 tests the hypothesis that transport involves translocation pathway conformational changes controlled by scaffold TM6 - pathway TM1 interactions. GLUT1 hydrogens (80%) undergo conformation-sensitive exchange with solvent tritium or deuterium but which amino acids exchange is unknown. We will identify them by H/DX-mass spectrometry of purified human GLUT1 trapped in inward, occluded and outward orientations. GLUT1-GLUT4 homology scanning mutagenesis shows that TM6 controls the rate of conformational change between inward and outward GLUT1 and GLUT4 orientations by interacting with TM1. We ask if this is a hallmark of all GLUTs, we investigate which TM6 residues interact with TM1 by tryptophan scanning and dicysteine cross-linking mutagenesis and ask if these residues are altered in GLUT1-DS. Specific Aim 3 tests the hypothesis that GLUT dimerization and tetramerization determine transport efficiency and are mediated by specific translocation and scaffold TMs. GLUT1 forms homo- dimers and homo-tetramers whereas GLUTs 2-4 form homo-dimers. The GLUTs do not hetero-oligomerize. Substituting GLUT1 scaffold TM9 into GLUT3 allows GLUT3 to tetramerize and to form heterocomplexes with GLUT1. GLUT3 TM9 causes GLUT1 to dissociate into dimers. Pathway TMs 5, 8, 2 and 11 may be responsible for isoform specific dimerization. We propose to test this hypothesis directly and outline experiments to investigate how GLUT activity is affected by oligomeric state and whether mutations that cause GLUT1-DS also affect GLUT1 quaternary structure and/or the effects of quaternary structure on function.